title: The N terminus of the murine p53 tumour suppressor is an independent regulatory domain affecting activation and thermostability.
abstract: The contribution of each of the structural domains of p53 to its function has been discussed widely in the literature. Crystallographic studies have revealed much about the structure of the core DNA binding domain, but as it has not been possible to use this approach for the intact protein, the effect of the domains flanking the core must be investigated by more indirect techniques. In this study a series of truncated murine p53 proteins has been investigated for DNA binding activity at 4 degrees C and 37 degrees C, transcriptional activation, and tumour suppression activity. Full-length p53, and truncations lacking the N terminus, purified from a baculovirus expression system all show latency for DNA binding; that is, they must be activated to bind by association with a C-terminal antibody such as PAb421. This demonstrates that latency for DNA binding is independent of the N terminus. Truncations lacking the C-terminal oligomerisation domain, and the isolated core domain, can only be activated to bind DNA and PAb1620 (an antibody recognising the wild-type conformation of the core domain) in the presence of cross-linking antibodies, while murine core only binds to DNA in the presence of PAb1620. An analysis of the thermostability of DNA binding revealed that antibodies that bind the N terminus of p53 could protect the protein against loss of activity at 37 degrees C. C-terminal antibodies, however, were ineffective unless the N-terminal 37 amino acid residues were absent. The N terminus may retain some secondary structure, since it is the main contributor to the anomalous migration in SDS-polyacrylamide gels. Our results suggest that the N terminus has a destabilising effect that influences conformation of p53 at 37 degrees C, so cellular proteins binding to the N terminus in vivo may modulate p53 conformation and stability. The effects on thermostability are also direct evidence showing that antibodies binding to N-terminal deletions create a conformational change in the rest of the molecule. In addition, longer deletions of the C terminus reduce the ability of p53 to transactivate target genes and inactivate tumour suppression activity, while truncations of the N terminus retain partial tumour suppression activity. Our results clearly show participation of both the N and C termini in the regulation of all the functions of p53 at 37 degrees C, indicating that distinct, independent domains interact with each other within, the flexible structure of p53.
authors.affiliation: Department of Biochemistry, University of Dundee, Scotland, UK.
authors.forename: S
authors.initials: S
authors.lastname: Hansen
authors.forename: D P
authors.initials: DP
authors.lastname: Lane
authors.forename: C A
authors.initials: CA
authors.lastname: Midgley
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53